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Anti nk1

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Anti-NK1.1 is a monoclonal antibody that binds to the NK1.1 antigen expressed on natural killer cells and certain T cell subsets in mice. It is commonly used as a tool for the detection and analysis of these cell populations in flow cytometry and other immunological applications.

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Lab products found in correlation

Anti cd3, by BioLegend (16 mentions) Anti cd4, by BioLegend (16 mentions) Anti cd45, by BioLegend (14 mentions) Anti cd8, by BioLegend (11 mentions) Anti cd19, by BioLegend (9 mentions) Anti cd11c, by BioLegend (9 mentions) Anti cd11b, by BioLegend (7 mentions) Anti ly6g, by BioLegend (7 mentions) Anti cd44, by BioLegend (6 mentions) Anti f4 80, by BioLegend (6 mentions) Anti ifn γ, by BioLegend (5 mentions) Anti cd62l, by BioLegend (5 mentions) Anti cd69, by BioLegend (5 mentions) Anti il 17a, by BioLegend (4 mentions) Anti tnf α, by BioLegend (4 mentions) Anti mhcii, by BioLegend (4 mentions) Anti nkp46, by BioLegend (4 mentions) Il 12, by Thermo Fisher Scientific (4 mentions) Anti b220, by BioLegend (4 mentions) Anti il 4, by BioLegend (3 mentions)

Spelling variants of this product

Anti-NK1.1 (clone PK136, (9 citations) Anti-NK1.1-FITC (6 citations) Anti NK1.1 (clone: PK (2 citations) BV650 anti-NK1.1 (2 citations) BV421 anti-mouse NK1.1 (2 citations) PE-cy7-conjugated anti-mouse NK1.1 (2 citations) Anti-NK1.1-BV650 (2 citations) FITC anti-mouse-NK1.1 (2 citations) PE/Cy7-anti-NK1.1 mAb (clone PK136, (2 citations) Anti-mouse NK1.1 (PK136) (2 citations)

33 protocols using anti nk1

1

Liver Leukocyte Isolation and Flow Cytometry

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Leukocytes were isolated from the liver in the way as described previously40 (link). For flow cytometry, cells were stained for surface antigens with anti-NK1.1, anti-CD3, anti-CD4, anti-CD8α, anti-CD19, anti-CD44, anti-CD62L (BioLegend, San Diego, CA), or/and for intracellular antigens with anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α and anti-IL-10 (BioLegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). Methods were the sameas those described previously9 (link).
Li Z., Zhang C., Li L., Bi X., Li L., Yang S., Zhang N., Wang H., Yang N., Abulizi A., Aini A., Lin R., Vuitton D.A, & Wen H. (2019). The local immune response during Echinococcus granulosus growth in a quantitative hepatic experimental model. Scientific Reports, 9, 19612.
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2

Comprehensive Immune Cell Profiling

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Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1: Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).
Metzger P., Kirchleitner S.V., Kluge M., Koenig L.M., Hörth C., Rambuscheck C.A., Böhmer D., Ahlfeld J., Kobold S., Friedel C.C., Endres S., Schnurr M, & Duewell P. (2019). Immunostimulatory RNA leads to functional reprogramming of myeloid-derived suppressor cells in pancreatic cancer. Journal for Immunotherapy of Cancer, 7, 288.
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3

IFN-γ Production Measurement Protocol

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For the measurement of IFN-γ production, splenocytes were activated in 96 well plates with hIL-15 (50 ng/ml), IL-12 (10 ng/ml, Peprotech), IL-18 (100 ng/ml), PMA (10 ng/ml), Ionomycin (1 μg/ml), or coated with anti-NK1.1 (25 μg/ml, BioLegend), anti-NKp46 (10 μg/ml, BioLegend), or LY49H (10 μg/ml, BioLegend). Cells were incubated with monensin and brefeldin A (BD GolgiPlug and GolgiStop) in complete medium for 4 h at 37°C. The cells were subjected to surface staining and intracellular staining was performed by use of the FoxP3/Transcription Factor Staining Buffer Set (eBioscience).
Delconte R.B., Guittard G., Goh W., Hediyeh-Zadeh S., Hennessy R.J., Rautela J., Davis M.J., Souza-Fonseca-Guimaraes F., Nunès J.A, & Huntington N.D. (2020). NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS. Frontiers in Immunology, 11, 75.
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4

Multiparameter Immune Cell Profiling

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Anti-mouse ABs: anti-CD3e (clone: 145-2C11), anti-CD34 (clone: HM34), anti-CD45R (clone: RA3-6B2), anti-CD117 (c-KIT, clone: 2B8), anti-CD182 (CXCR2, clone: SA044G4), anti-CD184 (CXCR4, clone: L276F12), anti-Ly-6A/E (Sca-1, clone: D7), anti-Ki-67 (clone: 11F6), anti-Ly-6G (clone: 1A8), anti-NK-1.1 (clone: PK1), anti-mouse/human-CD11b (clone: M1/70) all from BioLegend (San Diego, CA, USA), and anti-CD16/32 (clone: 93) and anti-CD62L (clone: MEL-14) (both from eBioscience, Thermo Fisher Scientific, Waltham, MA, USA). Anti-human ABs: anti-CD66b (clone: 80H3, Beckman Coulter, Brea, CA, USA), and anti-CD71 (clone: M-A712, BD Biosciences, Franklin Lakes, NJ, USA). Viability dye eFluor780 and eFluor506 (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) or DAPI (BioLegend, San Diego, CA, USA) were used to determine viable cells.
Siakaeva E., Pylaeva E., Spyra I., Bordbari S., Höing B., Kürten C., Lang S, & Jablonska J. (2019). Neutrophil Maturation and Survival Is Controlled by IFN-Dependent Regulation of NAMPT Signaling. International Journal of Molecular Sciences, 20(22), 5584.
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5

Isolation and Immunophenotyping of Tumor Cells

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Tumor tissues were minced and incubated with collagenase type II (1 mg/ml, Thermo Fisher) at 37°C for 40 min to obtain a single-cell suspension. For the staining of extracellular target proteins, 1 × 106 cells were first incubated in a mixture of PBS, 1% FBS, and anti-CD16/32 antibody (BioLegend, #101301) to block non-specific binding and then labeled with the indicated antibodies at room temperature for 30 min. Fluorescence-activated cell sorting (FACS) was performed using a Beckman Coulter Gallios flow cytometer (USA), and the results were analyzed using FlowJo software version 10.0.7 (TreeStar). The following fluorochrome-coupled antibodies were used in the experiment: anti-CD45 (#103115), anti-CD206 (#141711), anti-CD3 (#100235), anti-CD4 (#100203), anti-CD8 (#100733), anti-NK1.1 (#108709), and anti-Gr1 (#108425) (all from BioLegend); and anti-CD11b (#12-0112), anti-F4/80 (#11-4801), anti-CD11c (25–0114), anti-MHCII (#17-5321), and anti-CD86 (#17-0862) (all from eBioscience) antibodies; the fixable viability dye eFluor 506 was from eBioscience (#65-0866).
Yu H., Bai Y., Qiu J., He X., Xiong J., Dai Q., Wang X., Li Y., Sheng H., Xin R., Jiang L., Li Q., Li D., Zhang H., Zhang L., Chen Q., Peng J., Hu X, & Zhang K. (2021). Pseudomonas aeruginosa PcrV Enhances the Nitric Oxide-Mediated Tumoricidal Activity of Tumor-Associated Macrophages via a TLR4/PI3K/AKT/mTOR-Glycolysis-Nitric Oxide Circuit. Frontiers in Oncology, 11, 736882.
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6

Multiparameter Flow Cytometry Profiling

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Fluorochrome conjugated antibodies were purchased from Biolegend (anti-CD45 Cat# 103132, anti-CD4 Cat# 100546, anti-CD8 Cat# 100714, anti-NK1.1 Cat# 108710). Aqua fluorescent reactive dye (live/dead) was purchased from Invitrogen (Cat# L34966A), and Fc block was purchased from Biolegend (Cat# 101320) was performed. Flow cytometric analysis was performed on LSR II FACS analyzer (BD Biosciences) at the Saint Louis University Flow Cytometry Core Facility. Live lymphocytes were identified as staining negative for live/dead stain and positive for CD45. These were segregated into CD4+ and CD8+ T cells or CD4/8-negative NK1.1+ natural killer (NK) cells. CD4+ T cells were further segregated into conventional FoxP3-negative and Foxp3+ regulatory cells. Flow cytometry data was analyzed using FlowJo v.10 software (Tree Star Inc.).
Kuehm L.M., Khojandi N., Piening A., Klevorn L.E., Geraud S.C., McLaughlin N.R., Griffett K., Burris T.P., Pyles K.D., Nelson A.M., Preuss M.L., Bockerstett K.A., Donlin M.J., McCommis K.S., DiPaolo R.J, & Teague R.M. (2020). Fructose promotes cytoprotection in melanoma tumors and resistance to immunotherapy. Cancer immunology research, 9(2), 227-238.
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7

Multicolor Flow Cytometry Analysis

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EDTA-treated blood (500 µL) was lysed using red blood cell lysing buffer Hybri-Max (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the remaining leukocytes were labelled with the following antibodies (BioLegend, San Diego, CA, USA): anti-CD11c (N418), anti-I-Ab (KH74), anti-CD11b (M1/70), anti-CD3ε (145-2C11), anti-CD19 (6D5), anti-NK1.1 (PK136), anti-Ly-6C (HK1.4), and anti-Gr-1 (RB6-8C5). Labelling occurred in the dark at 4 °C in FACS buffer (PBS + 0.1% BSA (Sigma Aldrich, St. Louis, MO, USA) + 0.05% NaN3 (Merck, Kenilworth, NJ, USA)) containing CD16/32 Fc-receptor blocker (BioLegend, San Diego, CA, USA). Next, cells were analysed on a BD Accuri C6 cytometer equipped with a blue and red laser (Becton Dickinson, Erembodegem, Belgium). Dead cells were excluded based on forward scatter, side scatter and positive staining for propidium iodide (Invitrogen, Waltham, MA, USA). Data analysis was performed with FCS Express 4 (De Novo Software, Glendale, CA, USA).
Roth L., Rombouts M., Schrijvers D.M., Emini Veseli B., Martinet W, & De Meyer G.R. (2021). Acetylsalicylic Acid Reduces Passive Aortic Wall Stiffness and Cardiovascular Remodelling in a Mouse Model of Advanced Atherosclerosis. International Journal of Molecular Sciences, 23(1), 404.
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8

Detailed Antibody Characterization for Immunoblotting and Flow Cytometry

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Primary antibodies used for immunoblotting were: anti-Snrnp40 (HPA026527) from Atlas; anti-FLAG M2 (F1804) from Sigma-Aldrich; anti-Themis (06–1328) and anti-GAPDH (MAB374) from Millipore; anti-Snrnp200 (A303–453A-T) from Bethyl; anti-Eftud2 (10208–1-AP) from Proteintech; anti-Cd2bp2 (PA5–18286) from Invitrogen; anti-Ikzf3 (Aiolos, 15103), anti-IL-17A (13838), anti-IL-17F (13186), anti-α-tubulin (3873), anti-β-actin (3700), anti-Hsp90 (4874) and anti-GFP (2956) from Cell Signaling. Antibodies used for flow cytometry were: anti-CD3ε (145–2C11), anti-CD4 (RM4–5), anti-CD8α (53–6.7), anti-B220 (RA3–6B2), anti-NK-1.1 (PK136), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD11c (HL3), anti-CD11b (M1/70), anti-F4/80 (BM8), anti-mouse Lineage Cocktail (CD3/Ly-6G/CD11b/B220/Ter-119), anti-c-Kit (CD117, 2B8), anti-Sca-1 (Ly-6A/E, D7), anti-IL-7Rα (CD127, SB/199), anti-CD16/32 (93), anti-Flk-2 (CD135, A2F10), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD107a (Lamp-1, 1D4B), anti-TNF (MP6-XT22) from BioLegend or BD Biosciences, and anti-CD34 (RAM34), anti-granzyme B (NGZB), anti-IFN-γ (XMG1.2) from eBioscience.
Zhang D., Yue T., Choi J.H., Nair-Gill E., Zhong X., Wang K.W., Zhan X., Li X., Choi M., Tang M., Quan J., Hildebrand S., Moresco E.M, & Beutler B. (2019). Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40. Nature immunology, 20(10), 1322-1334.
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9

Immunofluorescence Staining of Liver Cryosections

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Liver cryosections (5 µm) were air-dried and fixed with ice-cold acetone. After rehydration in phosphate-buffered saline (PBS) the samples were treated with 2 N HCl for 30 min and afterwards neutralised with 0.1 M sodium borate (pH 8.0) for 9 min. The samples were washed in PBS. Antibodies were incubated in 0.2% bovine serum albumin (BSA) and 10% goat serum in PBS (anti-hAAT: 1∶200, RDI; anti-BrdU: 1∶40; Becton Dickinson, Heidelberg, Germany; anti-NK1,1: 1∶100; biolegend, Germany) for 1 h at 37°C or overnight at 4°C, respectively. For the detection of collagen tissue sections were fixed in 4% PFA (Roth, Karlsruhe, Germany) and blocked in 2% BSA in PBS acid. Incubation of the first antibody was performed overnight at 4°C or 1 h at room temperature, respectively (anti-collagen type I: 1∶250; Biotrend, Cologne, Germany; anti-Ki-67: 1∶100 in 0.3% Triton X-100, 5% goat serum in PBS; Nova Castra Laboratories, Newcastle upon Tyne, UK). After washing in PBS Alexa Fluor 488 and/or 594-conjugated secondary antibodies (Molecular Probes/Invitrogen, Karlsruhe, Germany) were used for immunofluorescence detection. The sections were analysed using a fluorescence microscope (Zeiss, Jena, Germany).
Kaldenbach M., Cubero F.J., Erschfeld S., Liedtke C., Trautwein C, & Streetz K. (2014). Hepatic Tissue Environment in NEMO-Deficient Mice Critically Regulates Positive Selection of Donor Cells after Hepatocyte Transplantation. PLoS ONE, 9(6), e100786.
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10

Murine Immune Cell Profiling

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A single cell suspension from thymi and spleens of 4–6 week-old mice was obtained using a Dounce homogenizer. Cells were first preincubated for 10 min on ice with Fc receptor-blocking anti-CD16/32 (clone 93) antibody in PBS-1% biotin-free BSA solution and then stained for 20 min with specific primary antibodies. Biotinylated primary antibodies were revealed with streptavidin conjugated fluorescent dyes (SAv-PE/Cy7, SAv-APC, SAv-APC/Cy7). Labeled cells were analyzed on BD FACSCantoII (BD Biosciences) or CytoFLEX (Beckman Coulter) flow cytometers and data analysis was performed using FlowJo software (FlowJo, LLC). Clones of monoclonal antibodies were: anti-CCR6 (29–2L17), anti-CD3ε (145–2C11), anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.7), anti-CD11c (N418), CD24 (M1/69), anti-CD25 (PC61), anti-CD27 (LG.3A10), anti-CD28 (E18), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 (RI7217), anti-CD73 (TY/11.8), anti-CD117 (c-Kit, 2B8) anti-B220 (RA3–6B2), anti-CD11b (Mac1, M1/70), anti-γδT-cell (GL3), hamster IgG-PE/Cy7 (HTK888), anti-Ly6G/Ly6C (GR1, RB6–8C5), anti-Nk1.1 (PK136), anti-Tcrβ (H57–597), anti-Tcrβ(Vα2) (B20.1), Ter-119 (all from Biolegend). Splenocytes were analyzed as previously described (18 (link)).
Zikmund T., Kokavec J., Turkova T., Savvulidi F., Paszekova H., Vodenkova S., Sedlacek R., Skoultchi A.I, & Stopka T. (2019). ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β-selection.. Journal of immunology (Baltimore, Md. : 1950), 202(12), 3434-3446.
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